A Double Strand Break during Telophase Is Repaired with Homologous Recombination Despite the Absence of an Available Sister Chromatid

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2421-2421
Author(s):  
Amit Patel ◽  
Luis Alcaide Aragon
Keyword(s):  
Homologous Recombination ◽  
Sister Chromatid ◽  
Conflicts Of Interest ◽  
Strand Break ◽  
Double Strand Break ◽  
Restriction Enzyme Digestion ◽  
Dsb Repair ◽  
Sister Chromatids ◽  
Kinetics Of ◽  
In Cells

Abstract Background: Chromosomal breakage results from a DNA double strand break (DSB), and is repaired to maintain and restore genetic integrity, principally through two major pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). HR is initiated by nucleolytic resection of a DSB in the presence of cyclin-dependent kinase 1 (Cdk1) activity. DSB repair through HR is dependent on Rad52, and can be error-free when a sister chromatid is used as a template for repair. However, HR is mutagenic when any other template is used for repair. Loss of nucleotides adjacent to the DSB is a feature of repair through NHEJ. There is co-relation between Cdk1 activity and the presence of a sister chromatid. The research question was, in addition to Cdk1 activity is the presence of an intact sister chromatid a requirement to initiate DSB repair with the HR pathway. Methods: Cdk1 activity peaks during mitosis in the presence of an intact sister chromatid. To study DSB resection and repair in cells arrested in either mitotic metaphase or telophase when Cdk1-Clb2 was active, conditional alleles were constructed in a eukaryotic haploid budding yeast model of HR. The model permitted simultaneous induction of a single site-specific DSB in cells that were synchronised to a phase of the cell division cycle. Physical monitoring of the kinetics of DSB formation, nucleolytic resection of adjacent DNA, and DSB repair, was achieved by probing Southern membranes after restriction enzyme digestion of extracted genomic DNA from time courses. Results: Sister chromatids were segregated during telophase arrest induced by either Cdc14 or Cdc15 depletion. Metaphase arrest was achieved with Cdc20 depletion, either directly, or indirectly by activation of the spindle assembly checkpoint by inhibition of microtubule polymerisation. Sister chromatids were unsegregated and physically attached through cohesin during metaphase. The absence of an intact sister chromatid did not prevent DSB repair with the HR pathway during telophase. Nucleolytic resection was observed in the presence or absence of an intra-chromosomal homologous but non-identical DNA repair template. The DSB cut site did not become resistant to cycles of re-cleavage through loss of adjacent nucleotides. DSB repair by HR was dependent on Rad52. The kinetics of nucleolytic resection adjacent to the DSB, and repair by HR, were similar during telophase and metaphase. Conclusions: This is the first study to report the observation that the availability of the sister chromatid is not a requirement to promote DSB repair with the HR pathway during telophase. Initiation of HR occurs despite segregated sister chromatids, even in the absence of a non-identical homologous DNA donor template, with inherently mutagenic repair by HR. This unexpected discovery has important clinical implications to the pathogenesis of chromosomal translocations and oncogenesis, and tumour progression with repair of treatment-induced DSBs. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Mre11-Rad50-Xrs2 Protein Complex Facilitates Homologous Recombination-Based Double-Strand Break Repair inSaccharomyces cerevisiae

1999 ◽  
Vol 19 (11) ◽  
pp. 7681-7687 ◽  
Author(s):  
Debra A. Bressan ◽  
Bonnie K. Baxter ◽  
John H. J. Petrini
Keyword(s):  
Homologous Recombination ◽  
Sister Chromatid ◽  
Protein Complex ◽  
Cellular Response ◽  
Strand Break ◽  
Double Strand Break ◽  
Dsb Repair ◽  
Synchronous Cultures ◽  
Dna Dsbs

ABSTRACT Saccharomyces cerevisiae mre11Δ mutants are profoundly deficient in double-strand break (DSB) repair, indicating that the Mre11-Rad50-Xrs2 protein complex plays a central role in the cellular response to DNA DSBs. In this study, we examined the role of the complex in homologous recombination, the primary mode of DSB repair in yeast. We measured survival in synchronous cultures following irradiation and scored sister chromatid and interhomologue recombination genetically. mre11Δ strains were profoundly sensitive to ionizing radiation (IR) throughout the cell cycle. Mutant strains exhibited decreased frequencies of IR-induced sister chromatid and interhomologue recombination, indicating a general deficiency in homologous recombination-based DSB repair. Since a nuclease-deficientmre11 mutant was not impaired in these assays, it appears that the role of the S. cerevisiae Mre11-Rad50-Xrs2 protein complex in facilitating homologous recombination is independent of its nuclease activities.

scholarly journals Genetic Requirements for RAD51- andRAD54-Independent Break-Induced Replication Repair of a Chromosomal Double-Strand Break

2001 ◽  
Vol 21 (6) ◽  
pp. 2048-2056 ◽  
Author(s):  
Laurence Signon ◽  
Anna Malkova ◽  
Maria L. Naylor ◽  
Hannah Klein ◽  
James E. Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Homologous Recombination ◽  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
Telomere Maintenance ◽  
Diploid Cells ◽  
Break Induced Replication ◽  
In Cells

ABSTRACT Broken chromosomes can be repaired by several homologous recombination mechanisms, including gene conversion and break-induced replication (BIR). In Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break (DSB) is normally repaired by gene conversion. Previously, we have shown that in the absence ofRAD52, repair is nearly absent and diploid cells lose the broken chromosome; however, in cells lacking RAD51, gene conversion is absent but cells can repair the DSB by BIR. We now report that gene conversion is also abolished when RAD54, RAD55, and RAD57 are deleted but BIR occurs, as withrad51Δ cells. DSB-induced gene conversion is not significantly affected when RAD50, RAD59, TID1(RDH54), SRS2, or SGS1 is deleted. Various double mutations largely eliminate both gene conversion and BIR, including rad51Δ rad50Δ, rad51Δ rad59Δ, andrad54Δ tid1Δ. These results demonstrate that there is aRAD51- and RAD54-independent BIR pathway that requires RAD59, TID1, RAD50, and presumablyMRE11 and XRS2. The similar genetic requirements for BIR and telomere maintenance in the absence of telomerase also suggest that these two processes proceed by similar mechanisms.

Double-strand-break-induced homologous recombination in mammalian cells

2001 ◽  
Vol 29 (2) ◽  
pp. 196-201 ◽  
Author(s):  
R. D. Johnson ◽  
M. Jasin
Keyword(s):  
Homologous Recombination ◽  
Sister Chromatid ◽  
Mammalian Cells ◽  
Indirect Evidence ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Crossing Over ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks

In mammalian cells, the repair of DNA double-strand breaks (DSBs) occurs by both homologous and non-homologous mechanisms. Indirect evidence, including that from gene targeting and random integration experiments, had suggested that non-homologous mechanisms were significantly more frequent than homologous ones. However, more recent experiments indicate that homologous recombination is also a prominent DSB repair pathway. These experiments show that mammalian cells use homologous sequences located at multiple positions throughout the genome to repair a DSB. However, template preference appears to be biased, with the sister chromatid being preferred by 2–3 orders of magnitude over a homologous or heterologous chromosome. The outcome of homologous recombination in mammalian cells is predominantly gene conversion that is not associated with crossing-over. The preference for the sister chromatid and the bias against crossing-over seen in mitotic mammalian cells may have developed in order to reduce the potential for genome alterations that could occur when other homologous repair templates are utilized. In attempts to understand further the mechanism of homologous recombination, the proteins that promote this process are beginning to be identified. To date, four mammalian proteins have been demonstrated conclusively to be involved in DSB repair by homologous recombination: Rad54, XRCC2, XRCC3 and BRCAI. This paper summarizes results from a number of recent studies.

scholarly journals The deSUMOylase SENP2 coordinates homologous recombination and non-homologous end joining by independent mechanisms

2018 ◽  
Author(s):  
Alexander J. Garvin ◽  
Alexandra K. Walker ◽  
Ruth M. Densham ◽  
Anoop Singh Chauhan ◽  
Helen R. Stone ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Strand Break ◽  
Double Strand Break ◽  
End Joining ◽  
Dsb Repair ◽  
Dna Double Strand Break ◽  
Non Homologous End Joining

AbstractSUMOylation in the DNA double-strand break (DSB) response regulates recruitment, activity and clearance of repair factors. However, our understanding of a role for deSUMOylation in this process is limited. Here we identify different mechanistic roles for deSUMOylation in homologous recombination (HR) and non-homologous enjoining (NHEJ) through the investigation of the deSUMOylase SENP2. We find regulated deSUMOylation of MDC1 prevents excessive SUMOylation and its RNF4-VCP mediated clearance from DSBs, thereby promoting NHEJ. In contrast we show HR is differentially sensitive to SUMO availability and SENP2 activity is needed to provide SUMO. SENP2 is amplified as part of the chromosome 3q amplification in many cancers. Increased SENP2 expression prolongs MDC1 foci retention and increases NHEJ and radioresistance. Collectively our data reveal that deSUMOylation differentially primes cells for responding to DSBs and demonstrates the ability of SENP2 to tune DSB repair responses.

scholarly journals Preferential localization of hyperphosphorylated replication protein A to double-strand break repair and checkpoint complexes upon DNA damage

2005 ◽  
Vol 391 (3) ◽  
pp. 473-480 ◽  
Author(s):  
Xiaoming Wu ◽  
Zhengguan Yang ◽  
Yiyong Liu ◽  
Yue Zou
Keyword(s):  
Dna Damage ◽  
Protein A ◽  
Replication Protein A ◽  
Strand Break ◽  
Double Strand Break ◽  
Replication Protein ◽  
Double Strand Break Repair ◽  
Dsb Repair ◽  
Checkpoint Activation ◽  
In Cells

RPA (replication protein A) is an essential factor for DNA DSB (double-strand break) repair and cell cycle checkpoint activation. The 32 kDa subunit of RPA undergoes hyperphosphorylation in response to cellular genotoxic insults. However, the potential involvement of hyperphosphorylated RPA in DSB repair and checkpoint activation remains unclear. Using co-immunoprecipitation assays, we showed that cellular interaction of RPA with two DSB repair factors, Rad51 and Rad52, was predominantly mediated by the hyperphosphorylated species of RPA in cells after UV and camptothecin treatment. Moreover, Rad51 and Rad52 displayed higher affinity for the hyperphosphorylated RPA than native RPA in an in vitro binding assay. Checkpoint kinase ATR (ataxia telangiectasia mutated and Rad3-related) also interacted more efficiently with the hyperphosphorylated RPA than with native RPA following DNA damage. Consistently, immunofluorescence microscopy demonstrated that the hyperphosphorylated RPA was able to co-localize with Rad52 and ATR to form significant nuclear foci in cells. Our results suggest that hyperphosphorylated RPA is preferentially localized to DSB repair and the DNA damage checkpoint complexes in response to DNA damage.

scholarly journals Accurate Homologous Recombination Is a Prominent Double-Strand Break Repair Pathway in Mammalian Chromosomes and Is Modulated by Mismatch Repair Protein Msh2

2007 ◽  
Vol 27 (22) ◽  
pp. 7816-7827 ◽  
Author(s):  
Jason A. Smith ◽  
Laura A. Bannister ◽  
Vikram Bhattacharjee ◽  
Yibin Wang ◽  
Barbara Criscuolo Waldman ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Cell Lines ◽  
Fusion Gene ◽  
Sequence Divergence ◽  
Strand Break ◽  
Double Strand Break ◽  
Repair Pathway ◽  
Dsb Repair ◽  
Spontaneous Recombination ◽  
Mammalian Cell Lines

ABSTRACT We designed DNA substrates to study intrachromosomal recombination in mammalian chromosomes. Each substrate contains a thymidine kinase (tk) gene fused to a neomycin resistance (neo) gene. The fusion gene is disrupted by an oligonucleotide containing the 18-bp recognition site for endonuclease I-SceI. Substrates also contain a “donor” tk sequence that displays 1% or 19% sequence divergence relative to the tk portion of the fusion gene. Each donor serves as a potential recombination partner for the fusion gene. After stably transfecting substrates into mammalian cell lines, we investigated spontaneous recombination and double-strand break (DSB)-induced recombination following I-SceI expression. No recombination events between sequences with 19% divergence were recovered. Strikingly, even though no selection for accurate repair was imposed, accurate conservative homologous recombination was the predominant DSB repair event recovered from rodent and human cell lines transfected with the substrate containing sequences displaying 1% divergence. Our work is the first unequivocal demonstration that homologous recombination can serve as a major DSB repair pathway in mammalian chromosomes. We also found that Msh2 can modulate homologous recombination in that Msh2 deficiency promoted discontinuity and increased length of gene conversion tracts and brought about a severalfold increase in the overall frequency of DSB-induced recombination.

scholarly journals Mouse RAD54 Affects DNA Double-Strand Break Repair and Sister Chromatid Exchange

2000 ◽  
Vol 20 (9) ◽  
pp. 3147-3156 ◽  
Author(s):  
Mies L. G. Dronkert ◽  
H. Berna Beverloo ◽  
Roger D. Johnson ◽  
Jan H. J. Hoeijmakers ◽  
Maria Jasin ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Gene Conversion ◽  
Sister Chromatid ◽  
Sister Chromatid Exchange ◽  
Embryonic Stem ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Single Strand Annealing ◽  
Strand Annealing

ABSTRACT Cells can achieve error-free repair of DNA double-strand breaks (DSBs) by homologous recombination through gene conversion with or without crossover. In contrast, an alternative homology-dependent DSB repair pathway, single-strand annealing (SSA), results in deletions. In this study, we analyzed the effect of mRAD54, a gene involved in homologous recombination, on the repair of a site-specific I-SceI-induced DSB located in a repeated DNA sequence in the genome of mouse embryonic stem cells. We used six isogenic cell lines differing solely in the orientation of the repeats. The combination of the three recombination-test substrates used discriminated among SSA, intrachromatid gene conversion, and sister chromatid gene conversion. DSB repair was most efficient for the substrate that allowed recovery of SSA events. Gene conversion with crossover, indistinguishable from long tract gene conversion, preferentially involved the sister chromatid rather than the repeat on the same chromatid. Comparing DSB repair in mRAD54wild-type and knockout cells revealed direct evidence for a role ofmRAD54 in DSB repair. The substrate measuring SSA showed an increased efficiency of DSB repair in the absence ofmRAD54. The substrate measuring sister chromatid gene conversion showed a decrease in gene conversion with and without crossover. Consistent with this observation, DNA damage-induced sister chromatid exchange was reduced in mRAD54-deficient cells. Our results suggest that mRAD54 promotes gene conversion with predominant use of the sister chromatid as the repair template at the expense of error-prone SSA.

scholarly journals The human HELLS chromatin remodelling protein promotes end resection to facilitate homologous recombination and contributes to DSB repair within heterochromatin

2019 ◽  
Vol 48 (4) ◽  
pp. 1872-1885 ◽  
Author(s):  
Gabriel Kollárovič ◽  
Caitríona E Topping ◽  
Edward P Shaw ◽  
Anna L Chambers
Keyword(s):  
Homologous Recombination ◽  
Cancer Biology ◽  
Genome Stability ◽  
Strand Break ◽  
Double Strand Break ◽  
Chromatin Remodelling ◽  
Double Strand Break Repair ◽  
Dsb Repair ◽  
Break Repair ◽  
End Resection

Abstract Efficient double-strand break repair in eukaryotes requires manipulation of chromatin structure. ATP-dependent chromatin remodelling enzymes facilitate different DNA repair pathways, during different stages of the cell cycle and in varied chromatin environments. The contribution of remodelling factors to double-strand break repair within heterochromatin during G2 is unclear. The human HELLS protein is a Snf2-like chromatin remodeller family member and is mutated or misregulated in several cancers and some cases of ICF syndrome. HELLS has been implicated in the DNA damage response, but its mechanistic function in repair is not well understood. We discover that HELLS facilitates homologous recombination at two-ended breaks and contributes to repair within heterochromatic regions during G2. HELLS promotes initiation of HR by facilitating end-resection and accumulation of CtIP at IR-induced foci. We identify an interaction between HELLS and CtIP and establish that the ATPase domain of HELLS is required to promote DSB repair. This function of HELLS in maintenance of genome stability is likely to contribute to its role in cancer biology and demonstrates that different chromatin remodelling activities are required for efficient repair in specific genomic contexts.

scholarly journals ZmRAD51C Is Essential for Double-Strand Break Repair and Homologous Recombination in Maize Meiosis

2019 ◽  
Vol 20 (21) ◽  
pp. 5513 ◽  
Author(s):  
Juli Jing ◽  
Ting Zhang ◽  
Yazhong Wang ◽  
Zhenhai Cui ◽  
Yan He
Keyword(s):  
Homologous Recombination ◽  
Early Stage ◽  
Central Element ◽  
Strand Break ◽  
Double Strand Break ◽  
Dsb Repair ◽  
Homologous Chromosomes ◽  
Meiotic Chromosomes ◽  
Homologous Chromosome Pairing ◽  
Axial Element

Radiation sensitive 51 (RAD51) recombinases play crucial roles in meiotic double-strand break (DSB) repair mediated by homologous recombination (HR) to ensure the correct segregation of homologous chromosomes. In this study, we identified the meiotic functions of ZmRAD51C, the maize homolog of Arabidopsis and rice RAD51C. The Zmrad51c mutants exhibited regular vegetative growth but complete sterility for both male and female inflorescence. However, the mutants showed hypersensitivity to DNA damage by mitomycin C. Cytological analysis indicated that homologous chromosome pairing and synapsis were rigorously inhibited, and meiotic chromosomes were often entangled from diplotene to metaphase I, leading to chromosome fragmentation at anaphase I. Immunofluorescence analysis showed that although the signals of the axial element absence of first division (AFD1) and asynaptic1 (ASY1) were normal, the assembly of the central element zipper1 (ZYP1) was severely disrupted. The DSB formation was normal in Zmrad51c meiocytes, symbolized by the regular occurrence of γH2AX signals. However, RAD51 and disrupted meiotic cDNA 1 (DMC1) signals were never detected at the early stage of prophase I in the mutant. Taken together, our results indicate that ZmRAD51C functions crucially for both meiotic DSB repair and homologous recombination in maize.

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